Method for Detection of Melanoma

ABSTRACT

The present invention provides non-invasive methods for detecting, monitoring, staging, and diagnosing malignant melanoma in a skin sample of a subject. The methods include analyzing expression in skin sample of one or more melanoma skin markers. The melanoma skin markers include IL-1 RI, endothelin-2, ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavy chain, Activin A (βA subunit), TNF RII, SPC4, and CNTF Rα. The skin sample can include nucleic acids, and can be a human skin sample from a lesion suspected of being melanoma.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/184,846 filed Jun. 27, 2002, now issued as U.S. Pat. No.7,297,480; which claims the benefit under 35 USC § 119(e) to U.S.Application Ser. No. 60/302,348 filed Jun. 28, 2001, now abandoned. Thedisclosure of each of the prior applications is considered part of andis incorporated by reference in the disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods related to melanoma patient fordetecting expression of genes in a skin sample of the epidermis relatedto malignant melanoma.

2. Background Information

Malignant melanoma ranks second among adult cancers (behind adultleukemia) in potential years of life lost. Each year, over 47,000 newcases are diagnosed, and the incidence of cutaneous melanoma appears tobe rising rapidly. Treatment of malignant melanoma involves surgicalexcision of the primary lesion, and vigilant monitoring to detectrecurrence. Currently, there is no approved therapy for patients havingintermediate risk of relapse. High-dose interferon, which can haveserious side effects, is approved for treatment of patients havinghigh-risk melanoma. There is no cure at this time for patients in whommetastasis to distant sites has occurred.

Dermatologists recommend that early detection of melanoma is the onlyway to reduce melanoma mortality by identifying curable lesions(Weinstock, JAMA 284:886-889, 2000). In practice, suspicious lesions arebiopsied or excised, and examined by histology. Clinical suspicion,however, largely depends on the experience and the skills of theexamining doctor. Very often the number of nevi under suspicion by farexceeds the number of lesions that could be removed. Erroneously excisedlesions are costly for the public and cause scars often in cosmeticallyimportant body regions. A non-invasive molecular tool for diagnosis istherefore desirable. Furthermore, even after a lesion is taken outdifferentiation of benign melanocytic lesions from melanomas can be verydifficult in a subset of about 10-15%, even for skilleddermatopathologists. If the diagnosis melanoma is made, tumor thicknessis among the most important factors to roughly evaluate the prognosis ofa melanoma patient (Breslow, Ann. Surg. 172:902-908, 1970). Molecularmarkers that could reflect the prognosis more precisely have also notbeen established. In addition, histological examination of cancer cellsdoes not adequately reflect the complicated series of molecular eventsunderlying the neoplastic process. Consequently, research efforts arenow being focused on molecular profiling of cancer cells using DNA arraytechnology, in which the activity of many genes or proteins are studiedin parallel. Molecular profiling of human cancer has been recentlyreviewed (Liotta and Petricoin, Nature Reviews/Genetics 1:48-56, 2000).

Several approaches have been taken to simplify the analysis of geneexpression profiles in tissue samples. Cell strains have been culturedto focus on a specific cell of interest. For example, some melanoma celllines appear to accurately represent the original primary material(Bittner et al 2000). However, cultured cells lack the regulatoryelements contributed by neighboring cells that affect gene expression invivo, such as cell-cell communication molecules, soluble factors, andextracellular matrix molecules.

More direct methods for gene expression profiling of cellular subtypesinclude the global survey approach and microdissection. In the globalapproach, the information content of interacting cells is preserved byextracting RNA directly from a heterogeneous piece of tissue. Tonormalize the data set for the actual abundance of normal tissue, inrelation to what is often minor amounts of diseased tissue, a referencegene set is constructed using RNA profiles extracted from a particularsubtype of cultured cells. Preservation of mRNA in tissue samples in theclinical setting is challenging, since RNA is very labile and issusceptible to abundant tissue RNases (Liotta and Petricoin, NatureReviews/Genetics 1:48-56, 2000).

An alternative approach, microdissection, utilizes mechanical force orlaser capture methods to select a cellular subtype from a tissue sample,for subsequent molecular profiling. Transition stages from normal cellsthrough carcinoma in situ, to invasive cancer can be identifiedmicroscopically, and profiled with microdissection techniques todiscover molecular events occurring along the progression to malignancy.A disadvantage to this approach is the level of expertise andinstrumentation required to select the pathological cells from thebiopsied tissue sample.

Therefore, there remains a need for effective methods to detectmalignant melanoma and to diagnose and stage melanoma from a suspiciousskin lesion.

SUMMARY OF THE INVENTION

The present specification discloses markers for early and late stagemalignant melanoma, and methods for detecting these markers usingnon-invasive sampling procedures. Accordingly, the present inventionprovides methods for detecting, staging, monitoring, diagnosing,prognosing and assisting in the management of malignant melanoma byanalyzing a skin sample for gene expression. For example, in one aspect,mRNAs of at least one of IL-1 RI, endothelin-2, ephrin-A5, IGF BindingProtein 7, HLA-A0202 heavy chain, Activin A (βA subunit), TNF RII, SPC4,and CNTF Rα are detected. In certain preferred embodiments, the skinsample is a human skin sample from a lesion suspected of being melanoma.

In one aspect, the present invention provides a non-invasive method fordetecting malignant melanoma in a skin sample of a subject, typically ahuman subject. The method includes analyzing expression in the skinsample, of one or more melanoma skin markers. The melanoma skin markersinclude, but are not limited to, IL-1 RI, endothelin-2, ephrin-A5, IGFBinding Protein 7, HLA-A0202 heavy chain, Activin A (βA subunit), TNFRII, SPC4, and CNTF Rα. The melanoma skin markers exhibit differentexpression levels in a skin sample at the site of a melanoma compared tolevels in a skin sample at the site of a benign lesion.

In one embodiment, the skin sample is obtained by applying at least oneapplication of an adhesive to the skin and removing the adhesive fromthe skin, or scraping the skin with an instrument to remove a samplecomprising a nucleic acid from the skin. Typically, nucleic acids,preferably ribonucleic acids (RNA), most preferably messenger RNA (mRNA)from the skin sample are analyzed.

In a preferred embodiment, the method includes analyzing nucleic acidsof the skin sample for expression levels of IL-1 RI, endothelin-2,ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavy chain, Activin A (βAsubunit), TNF RII, SPC4, and CNTF Rα. In this embodiment, expressionlevels of the IL-1 RI, endothelin-2, and ephrin-A5 are related to earlystage melanoma, and expression levels of IGF Binding Protein 7,HLA-A0202 heavy chain, Activin A (βA subunit), TNF RII, SPC4, and CNTFRα are related to late stage melanoma. More particularly, an increase inthe expression levels of IGF Binding Protein 7, HLA-A0202 heavy chain,Activin A (βA subunit), TNF RII, SPC4, and CNTF Rα are related to latestage melanoma. Furthermore, an increase in the expression levels ofendothelin-2, and ephrin-A5 genes are related to early stage melanoma.Finally, a decrease in levels of IL-1 RI are related to early stagemelanoma.

In another aspect, the present invention provides a non-invasive methodfor staging malignant melanoma in a subject skin sample. The methodincludes analyzing expression in a nucleic acid sample of one or moremelanoma skin markers. The melanoma skin markers include IL-1 RI,endothelin-2, ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavy chain,Activin A (βA subunit), TNF RII, SPC4, and CNTF Rα. Expression of themelanoma skin markers is related to melanoma.

In another aspect, the present invention provides a non-invasive methodfor staging malignant melanoma in a skin sample from a human subject.The method includes obtaining a skin sample by applying an adhesivesurface to the skin and removing the adhesive surface from the skin suchthat a skin sample comprising nucleic acid in an amount sufficient forsubsequent detection adheres to the adhesive surface after its removal.Then, analyzing expression of melanoma skin markers in nucleic acids ofthe skin sample. The melanoma skin markers include IL-1 RI,endothelin-2, ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavy chain,Activin A (βA subunit), TNF RII, SPC4, and CNTF Rα. In this aspect,expression of the IL-1 RI, endothelin-2, and ephrin-A5 is related toearly stage melanoma, and expression of IGF Binding Protein 7, HLA-A0202heavy chain, Activin A (βA subunit), TNF RII, SPC4, and CNTF Rα isrelated to late stage melanoma.

In another aspect, the present invention provides a non-invasive methodfor monitoring a suspicious lesion of a subject. The method includesanalyzing expression of one or more melanoma skin markers in a skinsample taken from the suspicious lesion at a first time point and asecond time point, and comparing the expression at the first time pointand the second time point. The melanoma skin markers include IL-1 RI,endothelin-2, ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavy chain,Activin A (βA subunit), TNF RII, SPC4, and CNTF Rα. Expression of themelanoma skin markers is related to melanoma, such that a change in theexpression of one or more of the melanoma skin markers over time isindicative of melanoma. In certain preferred embodiments, the skinsample includes nucleic acids, and is a human skin sample.

In another aspect, the present invention provides a non-invasive methodfor detecting expression of IL-1 RI, endothelin-2, ephrin-A5, IGFBinding Protein 7, HLA-A0202 heavy chain, Activin A (βA subunit), TNFRII, SPC4, and CNTF Rα genes in a skin sample. The method includesobtaining a skin sample by applying an adhesive surface to the skin andremoving the adhesive surface from the skin such that a skin samplecomprising nucleic acid in an amount sufficient for subsequent detectionadheres to the adhesive surface after its removal. Then, analyzingexpression of IL-1 RI, endothelin-2, ephrin-A5, IGF Binding Protein 7,HLA-A0202 heavy chain, Activin A (βA subunit), TNF RII, SPC4, and CNTFRα in the nucleic acids of the skin sample. Typically, for this aspectof the invention, mRNA from the skin sample are the analyzed nucleicacids.

In another aspect the present invention provides a kit for detectingmalignant melanoma. The kit includes one or more probes or primers thatselectively bind to one or more of Interleukin-1 RI (IL-1 RI),endothelin-2, ephrin-A5, Insulin-like Growth Factor (IGF) BindingProtein 7, Human Leukocyte Antigen (HLA)-A0202 heavy chain, Activin A(βA subunit), Tumor Necrosis Factor (TNF) RII, SPC4, and CiliaryNeurotrophic Factor (CNTF) Rα. The kit can include a skin samplecollection device, such as an adhesive strip.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an exposure of a gel representing the results for ribonucleaseprotection assay (RPA) performed with RNA obtained by tape strippingthree different areas of the upper arms of the same subject. Each of thethree sites were stripped 12 times. Four different RNA probes (IL-4,IL-8, L32, GADPH) were used for hybridization to RNA samples obtainedfrom the subject. Lane 1 shows the RNA isolated from an erythematousarea of skin, read clinically as 2+ erythema that was induced bysquarate (ACD). Shown in lane 3 is the RNA isolated from an ICDerythematous site (scored 2+) induced by 0.5% sodium lauryl sulfate(SLS). Both lanes demonstrate a band for IL-8. Lane 2 represents sampleobtained from non-inflamed, normal appearing skin of the same subject. Aband for the cytokine, IL-4, can be seen in lane 1 which was derivedfrom an allergic reaction.

FIG. 2 shows results for RPA performed with RNA obtained by tapestripping three different areas of the upper arm of four moreindividuals. Riboprobes for 6 different RNAs (IL-4, IL-8, IL-9, IL-13,IL-14 and an isoform of nitric oxide synthase (iNOS)) plus 2housekeeping genes were included in this gel. The “+” indicates that theskin harvested from the subject had been treated either with SLS (secondrow at bottom of figure) or squarate (third row at bottom of figure).

FIG. 3 shows quantitative analysis of expression of melanoma skinmarkers (as indicated under bars) in nevi versus advanced malignantmelanoma samples. Dark bars are expression in adhesive tape fromadvanced malignant melanoma. Light bars are expression in adhesive tapefrom melanocytes nevi (benign controls). Gene expression arbitrary unitsare based on phosphor imaging of array.

FIG. 4 shows quantitative analysis of expression of melanoma skinmarkers (as indicated under bars) in nevi versus early malignantmelanoma samples. Dark bars are expression in adhesive tape from earlymalignant melanoma. Light bars are expression in adhesive tape frommelanocytes nevi (benign controls). Gene expression arbitrary units arebased on phosphor imaging of array.

FIG. 5 shows semi quantitative real time PCR results for expression ofthe IL1 RI gene in nevus versus melanoma samples. Quantitation was basedon SyBr green incorporation values, normalized by GAPDH expression.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a non-invasive method fordetecting malignant melanoma in a skin sample of a subject. The methodincludes analyzing expression in skin sample of one or more melanomaskin markers. The melanoma skin markers include IL-1 RI, endothelin-2,ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavy chain, Activin A (βAsubunit), TNF RII, SPC4, and CNTF Rα. Expression of the melanoma skinmarkers is related to melanoma. In certain preferred embodiments, theskin sample includes nucleic acids, and is a human skin sample from alesion suspected of being melanoma.

In another aspect, the present invention provides a non-invasive methodfor staging malignant melanoma in a skin sample of a human subject. Themethod includes analyzing expression in the skin sample of one or moremelanoma skin markers. The melanoma skin markers include IL-1 RI,endothelin-2, ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavy chain,Activin A (βA subunit), TNF RII, SPC4, and CNTF Rα. Either an increaseor a decrease in expression of the melanoma skin markers, is related tomelanoma. In certain preferred embodiments, expression is analyzed byanalyzing mRNAs of one or more skin markers from the skin sample, and isa human skin sample from a lesion suspected of being melanoma.

In a preferred embodiment, the method includes analyzing expression inthe skin sample by measuring expression levels of IL-1 RI, endothelin-2,ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavy chain, Activin A (βAsubunit), TNF RII, SPC4, and CNTF Rα mRNAs. Expression levels of theIL-1 RI, endothelin-2, and ephrin-A5 genes are related to early stagemelanoma, and expression levels of the genes for IGF Binding Protein 7,HLA-A0202 heavy chain, Activin A (βA subunit), TNF RII, SPC4, and CNTFRα are related to late stage melanoma. More particularly, an increase inthe expression levels of IGF Binding Protein 7, HLA-A0202 heavy chain,Activin A (βA subunit), TNF RII, SPC4, and CNTF Rα are related to latestage melanoma. Furthermore, an increase in the expression levels ofendothelin-2, and ephrin-A5 genes are related to early stage melanoma.Finally, a decrease in levels of IL-1 RI are related to early stagemelanoma. The method can include the combination of all of the melanomaskin markers, or all of the early stage melanoma skin markers, or all ofthe late-stage melanoma skin markers, weighted according to F-power in amultivariate analysis, to increase the accuracy of the method.

In another preferred embodiment, the method includes detectingexpression of IL-1 RI, endothelin-2, and ephrin-A5 to detect malignantmelanoma in patients at risk for developing malignant melanoma. Thesegenes are identified herein as being differentially expressed inearly-stage melanoma. In another preferred embodiment, the methodincludes detecting expression of endothelin-2 and ephrin-A5.

The method for detecting malignant melanoma, may be a moleculardiagnostic screening test for early melanoma that includes analyzingmRNAs of the markers IL-1 RI, endothelin-2, and ephrin-A5 in nucleicacids, typically RNA extracted from a skin sample of a patientpopulation, such as those at risk for developing malignant melanoma dueto a family history of malignant melanoma. The sample is preferablyobtained using a non-invasive skin surface sampling method, as describedherein. The methods for detecting malignant melanoma of the presentinvention, can serve as aids for initial therapeutic decisions, savingmany lives and reducing the cost of this safety by avoiding unnecessaryexcisions and biopsies.

The methods of the present invention are particularly useful in thefamily practice setting for managing patients at risk for developingmelanoma. For example, the methods are useful in the following patientgroups: a) patients with multiple moles, especially when excisions ofall suspected moles can not be performed or suspicious lesions reside incosmetically problematical body parts, e.g. the face, the breast, anddécolleté in females etc.; b) patients with dysplastic nevus syndrome athigher risk to get melanoma, c) patients who have suffered from primarymelanoma, and therefore are at increased risk to get a second one; andd) patients with familial risk (i.e. genetic predisposition) formelanoma.

The methods of the present invention use a non-invasive method to obtaina sample of skin from a subject suspected of having melanoma or atincreased risk of developing melanoma. In certain preferred embodiments,the skin sample is a human skin sample from a lesion suspected of beingmelanoma. In preferred embodiments, the skin sample includes nucleicacids, which can then be analyzed using methods such as, but not limitedto, those described below. Levels of melanoma skin markers can bequantitated in the sample by measuring their absolute or relativeexpression at the protein or preferably RNA levels, and comparing theselevels to those of normal, control samples. As illustrated in Example 4herein, information regarding expression of malignant melanoma skinmarkers disclosed herein, provides information regarding the presenceand the stage of malignant melanoma. A decrease in expression levels ofIL-1 RI and/or an increase in expression levels of endothelin-2 and/orephrin-A5 is indicative of early stage melanoma. An increase inexpression levels of IGF Binding Protein 7, HLA-A0202 heavy chain,Activin A (βA subunit), TNF RII, SPC4, and/or CNTF Rα is indicative oflate stage melanoma.

It will be recognized that the non-invasive sample is typically takenfrom below the stratum corneum of the skin. As illustrated in Example 4,by using the methods of the invention it is possible to detect and stagemalignant melanoma by analyzing the melanoma skin markers disclosedherein.

In one embodiment, expression is analyzed for at least one of IL-1 RI,endothelin-2, and ephrin-A5. In another embodiment, expression isanalyzed for at least two, or all, of IL-1 RI, endothelin-2, andephrin-A5. In another embodiment, expression is analyzed for one or bothof endothelin-2 and ephrin-A5. In another embodiment, expression isanalyzed for at least one of IGF Binding Protein 7, HLA-A0202 heavychain, Activin A (□A subunit), TNF RII, SPC4, and CNTF R {tilde over(□)} In another embodiment, expression is analyzed for IGF BindingProtein 7, HLA-A0202 heavy chain, Activin A (□A subunit), TNF RII, SPC4,and CNTF R{tilde over (□)} In another embodiment, expression is analyzedfor at least one of IGF Binding Protein 7, HLA-A0202 heavy chain,Activin A (□A subunit), TNF RII, SPC4, and CNTF R{tilde over (□)} Inanother embodiment, expression is analyzed for at least one ofendothelin-1, ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavy chain,Activin A (□A subunit), TNF RII, SPC4, and CNTF R{tilde over (□)}

A “melanoma skin marker” is a gene whose expression level is differentbetween skin surface samples at the site of malignant melanoma and skinsurface samples of normal skin or a lesion, which is benign, such as abenign nevus. Therefore, expression of a melanoma skin marker is relatedto, or indicative of, melanoma. As discussed herein, all of the melanomaskin markers of the present invention exhibit increased expression inmelanoma cells as compared to benign nevi, except for IL1 RI. Manystatistical techniques are known in the art, which can be used todetermine whether a statistically significant difference in expressionis observed at a 90% or preferably a 95% confidence level. Example 4illustrates the use of a statistical test to identify melanoma skinmarkers. An increase or decrease in expression of these genes is relatedto malignant melanoma. In certain preferred embodiments, there is atleast a two-fold difference in levels between skin sample near the siteof malignant melanoma and skin samples from normal skin.

Melanoma skin markers identified herein include Interleukin-1 Receptor(IL-1 RI) (Genbank accession number X16896, Human mRNA for interleukin-1receptor); Endothelin-2 (Genbank accession number M65199, Humanendothelin 2 (ET2) mRNA); Ephrin-A5 (Genbank accession number U26403,Human receptor tyrosine kinase ligand LERK-7 precursor (EPLG7) mRNA);Insulin-like Growth Factor (IGF) Binding Protein 7 (Genbank accessionnumber L19182, Human MAC25 mRNA); Human Leukocyte Antigen (HLA)-A0202heavy chain (Genbank accession number M84379, Human MHC class Ilymphocyte antigen (HLA-A 0201) mRNA); Activin A (βA subunit) (Genbankaccession number M13436, Human ovarian beta-A inhibin mRNA); TumorNecrosis Factor (TNF) RII (Genbank accession number M55994, Human tumornecrosis factor receptor II (TNFrII) mRNA); SPC4 (Genbank accessionnumber M80482, Human subtilisin-like protein (PACE4) mRNA); and CiliaryNeurotrophic Factor (CNTF) Rα (Genbank accession number M73238, Humanciliary neurotrohic factor receptor (CNTFR) mRNA). One of ordinary skillin the art recognizes that Genbank accession numbers refer to numbersused to identify nucleotide sequences available in Genbank (Benson etal., “GenBank,” Nucleic Acids. Res. 30(1):17-20 (2002)).

As used herein, the terms “nucleic acid,” “polynucleotide,” or “nucleicacid sequence” refer to a polymer of deoxyribonucleotides orribonucleotides, in the form of a separate fragment or as a component ofa larger construct. Polynucleotide or nucleic acid sequences of theinvention include DNA, RNA, including mRNA and cDNA sequences. Thepolynucleotides of the sample of the present invention are typicallyRNA.

As used herein, the term “polypeptide” refers to a polymer of amino acidresidues in the form of a separate fragment or component of a largerconstruct. An example of a polypeptide includes amino acid sequencesencoding a cytokine or fragments thereof. A polypeptide may encode for afunctional protein or fragments of a protein. For example, anendothelin-2 polypeptide includes the full-length protein sequence ofendothelin-2 as well as fragments thereof consisting of a polymer ofamino acids.

In a preferred embodiment, the skin sample is obtained by applying atleast one application of an adhesive to the skin and removing theadhesive from the skin, or by scraping the skin with an instrument toremove a sample comprising a nucleic acid from the skin. The skin samplecan be obtained by using a tape stripping methodology in which one ormore tape strips are applied to the same skin site. Methods fornon-invasively obtaining a skin sample are discussed in U.S. patentapplication Ser. No. 09/375,609, filed Aug. 17, 1999, incorporatedherein by reference.

The skin sample can be obtained by applying the adhesive surface to theskin between 1 and 50 times, preferably between 1 and 25 times. Incertain preferred embodiments, the adhesive surface is applied to theskin between 1 and 3, most preferably between 1 and 2 times, to obtainthe skin sample. Using a preferred non-invasive sampling method, such asa tape-stripping methodology, the sample is obtained in a manner suchthat the skin nucleic acid profile after application is not affected forup to about one hour, and typically for up to two hours.

Skin samples obtained on adhesive films can be frozen before beinganalyzed using the methods of the present invention. Typically, this isperformed by snap-freezing a sample, as illustrated in Example 4, usingliquid nitrogen or dry ice.

The term “skin” means a tissue comprising a sheet of cells, one orseveral layers thick, organized above a basal lamina, and oftenspecialized for mechanical protection or active transport. In apreferred embodiment, the skin is mammalian skin. In a more preferredembodiment the skin is human skin.

The epidermis of the human skin comprises several distinct layers ofskin tissue. The deepest layer is the stratum basalis layer, whichconsists of columnar cells. The overlying layer is the stratum spinosum,which is composed of polyhedral cells. Cells pushed up from the stratumspinosum are flattened and synthesize keratohyalin granules to form thestratum granulosum layer. As these cells move outward, they lose theirnuclei, and the keratohyalin granules fuse and mingle with tonofibrils.This forms a clear layer called the stratum lucidum. The cells of thestratum lucidum are closely packed. As the cells move up from thestratum lucidum, they become compressed into many layers of opaquesquamae. These cells are all flattened remnants of cells that havebecome completely filled with keratin and have lost all other internalstructure, including nuclei. These squamae constitute the outer layer ofthe epidermis, the stratum corneum. At the bottom of the stratumcorneum, the cells are closely compacted and adhere to each otherstrongly, but higher in the stratum they become loosely packed, andeventually flake away at the surface.

In preferred embodiments of the present invention, the skin sampleincludes epidermal cells. The epidermis consists predominantly ofkeratinocytes (>90%), which differentiate from the basal layer, movingoutward through various layers having decreasing levels of cellularorganization, to become the cornified cells of the stratum corneumlayer. Renewal of the epidermis occurs every 20-30 days in normal skin.Other cell types present in the epidermis include melanocytes,Langerhans cells, and Merkel cells. Preferably, the skin sample of themethods of the present invention is an epidermis skin sample.

The term “sample” refers to any preparation derived from skin of asubject. For example, a sample of cells obtained using the non-invasivemethod described above may be used to isolate polynucleotides,polypeptides, or lipids, preferably polynucleotides and polypeptides,most preferably polynucleotides, for the methods of the presentinvention. In addition, the methods of the invention can be used invitro, for example with skin cells cultured on a solid or semi-solidsupport and organotypic skin constructs. In such instances, the skincells may be from any source suspected of being melanoma or from anindividual at risk of developing melanoma.

Samples for the present invention, typically are taken from a mole, orfrom another type of suspicious lesion (i.e. lesion suspected of beingmelanoma), especially one that resides in cosmetically problematicalbody parts, e.g. the face, the breast, décolleté in females, etc. Thesamples are taken of the skin surface of the suspicious lesion usingnon-invasive skin sampling methods discussed herein.

A “skin lesion” is a change in the color or texture in an area of skin.“Skin lesions suspected of being melanoma” are skin lesions withcharacteristics of malignant melanoma, which are well known todermatologists and oncologists. Such lesions are sometimes raised andcan have a color that is different from the color of normal skin of anindividual (e.g. brown, black, red, or bluish). Lesions suspected ofbeing melanoma sometimes include a mixture of colors, are oftenasymmetrical, can change in appearance over time, and may bleed. A skinlesion suspected of being melanoma may be a mole or nevus. Melanomalesions are usually, but not always, larger than 6 mm in diameter.Melanoma includes superficial spreading melanoma, nodular melanoma,acral lentiginous melanoma, and lentigo-maligna melanoma. Melanoma canoccur on skin that has been overexposed to the sun. Therefore, in oneembodiment the skin sample is taken from an area of skin that has beenoverexposed to the sun.

Typically, prior to the present invention a skin biopsy and histologicalexamination was used to confirm diagnosis of a lesion as malignantmelanoma. Furthermore, an X-ray, CT scan, MRI, or other procedures aresometimes indicated to determine if spreading (metastasis) has occurred.Such methods could be used in conjunction with the present invention, orcould be eliminated as a result of the present invention.

The methods of the present invention which detect the melanoma skinmarkers identified herein have utility not only in detecting and staginga skin sample from a mole or other lesion suspected of being melanoma,but also in diagnosing, and prognosing malignant melanoma.

Samples from a tissue can be isolated by any number of means well knownin the art. Invasive methods for isolating a sample include the use ofneedles, for example during blood sampling, as well as biopsies ofvarious tissues. Due to the invasive nature of these techniques there isan increased risk of mortality and morbidity. The methods and kits ofthe present invention typically use a non-invasive sampling method toobtain a skin sample.

Methods of the present invention can include a rapid, non-invasiveskin-sampling method for obtaining polynucleotides, typically RNA,preferably mRNA. The process of tape stripping itself has been shown notto affect the skin cytokine profile during the first two hours after theprocedure is done.

A non-invasive sampling method can include scraping epidermal cells ofthe skin with a rigid instrument. The instrument can be, for example, asterile #15 scalpel. However, it will be recognized that any number ofrigid instruments capable of removing only the surface layer (i.e.,stratum corneum) of the skin may be used. Alternatively, instead ofscraping the skin, the skin's epidermal layer may be removed by using anadhesive surface, such as, but not limited to an adhesive tape, forexample, but not limited to, Duct tape (333 Duct tape, Nashua tapeproducts), Scotch® tape (3M Scotch 810, St. Paul, Minn.), or a similaradhesive product. A preferred, but non-limiting example of an adhesivetape is D-SQUAME® (CuDerm, Dallas, Tex.).

In this embodiment the skin is stripped with the tape and the strippedcells and cellular material are then recovered from the scalpel, tape orother item. For example, tape used to obtain skin cells and cellularmaterial can be centrifuged in a sterile microfuge tube containing lysisbuffer. In the case of the scalpel the cells and cellular material canbe transferred to a sterile Petri dish and any cells present lysedtherein with lysis buffer. The same lysis buffer may be reused for eachpiece of tape or scalpel used at a single skin site. For certainapplications, the tape stripping method can be combined with thescraping method for removing cells and cellular material from the skin.The sample obtained can then be further processed, for example toisolate nucleic acids, polypeptides, or lipids. Preferably, the methodutilized does not adversely affect the polynucleotide, polypeptide, orlipid level being measured.

Polynucleotides can be isolated from the lysed cells and cellularmaterial by any number of means well known to those skilled in the art.For example, a number of commercial products are available for isolatingpolynucleotides, including but not limited to, TriReagent (MolecularResearch Center, Inc, Cincinnati, Ohio) can be used. The isolatedpolynucleotides can then be tested or assayed for particular nucleicacid sequences, including a polynucleotide encoding a cytokine.

Expression of melanoma skin markers is analyzed in the methods of thepresent invention. Analyzing expression includes any qualitative orquantitative method for detecting expression of a gene, many of whichare known in the art. The method can include analyzing expression of themelanoma skin markers by measuring expression of the melanoma skinmarkers using a quantitative method, or by using a qualitative method.Non-limiting methods for analyzing polynucleotides and polypeptides arediscussed below. Preferably, expression is analyzed using methods thatare directed to polynucleotides.

The methods of analyzing expression of a malignant melanoma of thepresent invention can utilize a biochip, or other miniaturehigh-throughput technology, for detecting expression of two or moremalignant melanoma skin markers. As illustrated in Example 4, themanufacture and use of biochips such as those involving bioarrays, areknown in the art and commercially available (See e.g., bioarraysavailable from Sigma-Genosys (The Woodlands, Tex.); Affymetrix (SantaClara, Calif.), and Full Moon Biosystems (Sunnyvale, Calif.)) (Forreviews of Biochips and bioarrays see, e.g., Kallioniemi O. P., “Biochiptechnologies in cancer research,” Ann Med, March; 33(2):142-7 (2001);and Rudert F., “Genomics and proteomics tools for the clinic,” CurrOpin. Mol. Ther., December; 2(6):633-42 (2000)).

Such bioarrays can be analyzed using blotting techniques similar tothose discussed below for conventional techniques of detectingpolynucleotides and polypeptides, as illustrated in Example 4. Othermicrofluidic devices and methods for analyzing gene expression,especially those in which more than one gene can be analyzedsimultaneously and those involving high-throughput technologies, can beused for the methods of the present invention.

Quantitative measurement of expression levels using bioarrays is alsoknown in the art, and typically involve a modified version of atraditional method for measuring expression as described herein. Forexample, such quantitation can be performed by measuring a phosphorimage of a radioactive-labeled probe binding to a spot of a microarray,using a phosphohor imager and imaging software.

The method of the present invention typically employ RNA, includingmessenger RNA (mRNA), isolated from a skin sample. The RNA may be singlestranded or double stranded. Enzymes and conditions optimal for reversetranscribing the template to DNA well known in the art can be used.Alternatively, the RNA can be subjected to RNAse protection assays. ADNA-RNA hybrid that contains one strand of each can also be used. Amixture of polynucleotides can also be employed, or the polynucleotidesproduced in a previous amplification reaction, using the same ordifferent primers may be so used. In the instance where thepolynucleotide sequence is to be amplified the polynucleotide sequencemay be a fraction of a melanoma skin marker, or can be present initiallyas a discrete molecule, such that the specific sequence is the entirenucleic acid. It is not necessary that the sequence to be amplified bepresent initially in a pure form; it may be a minor fraction of acomplex mixture.

In addition, RNAse protection assays can be used if RNA is thepolynucleotide obtained from the sample. In this procedure, a labeledantisense RNA probe is hybridized to the complementary polynucleotide inthe sample. The remaining unhybridized single-stranded probe is degradedby ribonuclease treatment. The hybridized, double stranded probe isprotected from RNAse digestion. After an appropriate time, the productsof the digestion reaction are collected and analyzed on a gel (see forexample Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, section4.7.1 (1987)). As used herein, “RNA probe” refers to a ribonucleotidecapable of hybridizing to RNA in a sample of interest. Those skilled inthe art will be able to identify and modify the RNAse protection assayspecific to the polynucleotide to be measured, for example, probespecificity may be altered, hybridization temperatures, quantity ofnucleic acid etc. Additionally, a number of commercial kits areavailable, for example, RiboQuant™ Multi-Probe RNAse Protection AssaySystem (Pharmingen, Inc., San Diego, Calif.).

In another embodiment, the polynucleotide in the sample may be analyzedby a blotting procedure, typically a Northern blot procedure. Forblotting procedures polynucleotides are separated on a gel and thenprobed with a complementary polynucleotide to the sequence of interest.For example, RNA is separated on a gel transferred to nitrocellulose andprobed with complementary DNA to one of the melanomadifferentially-diagnosed genes disclosed herein. The complementary probemay be labeled radioactively, chemically etc. Hybridization of the probeis indicative of the presence of the melanoma of interest.

Detection of a polynucleotide encoding a melanoma skin marker can beperformed by standard methods such as size fractionating the nucleicacid. Methods of size fractionating the DNA and RNA are well known tothose of skill in the art, such as by gel electrophoresis, includingpolyacrylamide gel electrophoresis (PAGE). For example, the gel may be adenaturing 7 M or 8 M urea-polyacrylamide-formamide gel. Sizefractionating the nucleic acid may also be accomplished bychromatographic methods known to those of skill in the art.

The detection of polynucleotides may optionally be performed by usingradioactively labeled probes. Any radioactive label may be employedwhich provides an adequate signal. Other labels include ligands, coloreddyes, and fluorescent molecules, which can serve as a specific bindingpair member for a labeled ligand, and the like. The labeled preparationsare used to probe for a polynucleotide by the Southern or Northernhybridization techniques, for example. Nucleotides obtained from samplesare transferred to filters that bind polynucleotides. After exposure tothe labeled polynucleotide probe, which will hybridize to nucleotidefragments containing target nucleic acid sequences, the binding of theradioactive probe to target nucleic acid fragments is identified byautoradiography (see Genetic Engineering, 1 ed. Robert Williamson,Academic Press (1981), pp. 72-81). The particular hybridizationtechnique is not essential to the invention. Hybridization techniquesare well known or easily ascertained by one of ordinary skill in theart. As improvements are made in hybridization techniques, they canreadily be applied in the method of the invention.

Probes according to the present invention and used in a method of thepresent invention selectively hybridize to the melanoma skin markersdisclosed herein. In preferred embodiments, the probes are spotted on abioarray using methods known in the art. As used herein, the term“selective hybridization” or “selectively hybridize,” refers tohybridization under moderately stringent or highly stringent conditionssuch that a nucleotide sequence preferentially associates with aselected nucleotide sequence over unrelated nucleotide sequences to alarge enough extent to be useful in detecting expression of a melanomaskin marker. It will be recognized that some amount of non-specifichybridization is unavoidable, but is acceptable provide thathybridization to a target nucleotide sequence is sufficiently selectivesuch that it can be distinguished over the non-specificcross-hybridization, for example, at least about 2-fold more selective,generally at least about 3-fold more selective, usually at least about5-fold more selective, and particularly at least about 10-fold moreselective, as determined, for example, by an amount of labeledoligonucleotide that binds to target nucleic acid molecule as comparedto a nucleic acid molecule other than the target molecule, particularlya substantially similar (i.e., homologous) nucleic acid molecule otherthan the target nucleic acid molecule.

Conditions that allow for selective hybridization can be determinedempirically, or can be estimated based, for example, on the relativeGC:AT content of the hybridizing oligonucleotide and the sequence towhich it is to hybridize, the length of the hybridizing oligonucleotide,and the number, if any, of mismatches between the oligonucleotide andsequence to which it is to hybridize (see, for example, Sambrook et al.,“Molecular Cloning: A laboratory manual (Cold Spring Harbor LaboratoryPress 1989)). An example of progressively higher stringency conditionsis as follows: 2×SSC/0.1% SDS at about room temperature (hybridizationconditions); 0.2×SSC/0.1% SDS at about room temperature (low stringencyconditions); 0.2×SSC/0.1% SDS at about 42EC (moderate stringencyconditions); and 0.1×SSC at about 68EC (high stringency conditions).Washing can be carried out using only one of these conditions, e.g.,high stringency conditions, or each of the conditions can be used, e.g.,for 10-15 minutes each, in the order listed above, repeating any or allof the steps listed. However, as mentioned above, optimal conditionswill vary, depending on the particular hybridization reaction involved,and can be determined empirically.

The polynucleotides encoding melanoma skin markers may be amplifiedbefore they are detected. The term “amplified” refers to the process ofmaking multiple copies of the nucleic acid from a single polynucleotidemolecule. The amplification of polynucleotides can be carried out invitro by biochemical processes known to those of skill in the art. Theamplification agent may be any compound or system that will function toaccomplish the synthesis of primer extension products, includingenzymes. Suitable enzymes for this purpose include, for example, E. coliDNA polymerase I, Taq polymerase, Klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, other available DNA polymerases,polymerase muteins, reverse transcriptase, ligase, and other enzymes,including heat-stable enzymes (i.e., those enzymes that perform primerextension after being subjected to temperatures sufficiently elevated tocause denaturation). Suitable enzymes will facilitate combination of thenucleotides in the proper manner to form the primer extension productsthat are complementary to each mutant nucleotide strand. Generally, thesynthesis will be initiated at the 3′-end of each primer and proceed inthe 5′-direction along the template strand, until synthesis terminates,producing molecules of different lengths. There may be amplificationagents, however, that initiate synthesis at the 5′-end and proceed inthe other direction, using the same process as described above. In anyevent, the method of the invention is not to be limited to theembodiments of amplification described herein.

One method of in vitro amplification, which can be used according tothis invention, is the polymerase chain reaction (PCR) described in U.S.Pat. Nos. 4,683,202 and 4,683,195. The term “polymerase chain reaction”refers to a method for amplifying a DNA base sequence using aheat-stable DNA polymerase and two oligonucleotide primers, onecomplementary to the (+)-strand at one end of the sequence to beamplified and the other complementary to the (−)-strand at the otherend. Because the newly synthesized DNA strands can subsequently serve asadditional templates for the same primer sequences, successive rounds ofprimer annealing, strand elongation, and dissociation produce rapid andhighly specific amplification of the desired sequence. The polymerasechain reaction is used to detect the presence of polynucleotidesencoding cytokines in the sample. Many polymerase chain methods areknown to those of skill in the art and may be used in the method of theinvention. For example, DNA can be subjected to 30 to 35 cycles ofamplification in a thermocycler as follows: 95° C. for 30 sec, 52° to60° C. for 1 min, and 72° C. for 1 min, with a final extension step of72° C. for 5 min. For another example, DNA can be subjected to 35polymerase chain reaction cycles in a thermocycler at a denaturingtemperature of 95° C. for 30 sec, followed by varying annealingtemperatures ranging from 54-58° C. for 1 min, an extension step at 70°C. for 1 min and a final extension step at 70° C.

The primers for use in amplifying the polynucleotides of the inventionmay be prepared using any suitable method, such as conventionalphosphotriester and phosphodiester methods or automated embodimentsthereof so long as the primers are capable of hybridizing to thepolynucleotides of interest. One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. The exact length of primer will depend on many factors,including temperature, buffer, and nucleotide composition. The primermust prime the synthesis of extension products in the presence of theinducing agent for amplification.

Primers used according to the method of the invention are complementaryto each strand of nucleotide sequence to be amplified. The term“complementary” means that the primers must hybridize with theirrespective strands under conditions, which allow the agent forpolymerization to function. In other words, the primers that arecomplementary to the flanking sequences hybridize with the flankingsequences and permit amplification of the nucleotide sequence.Preferably, the 3′ terminus of the primer that is extended has perfectlybase paired complementarity with the complementary flanking strand.

Primers and probes for the differentially expressed melanoma skinmarkers of the present invention, can be developed using known methodscombined with the present disclosure. For example, but not intended tobe limiting, PCR primers for IL1 RI can include:

Forward: TTCAGGACATTACTATTGCG (SEQ ID NO:1) (Target CGCAATAGTAATGTCCTGAA(SEQ ID NO:2) Reverse: TTCCACACTGTAATAGTCTTC (SEQ ID NO:3) (TargetGAAGACTATTACAGTGTGGAA (SEQ ID NO:4)

Another non-limiting example of primers and probes of the presentinvention, are primers and probes that selectively hybridize to thetarget sequences identified above (SEQ ID NOS: 2 and 4).

Those of ordinary skill in the art will know of various amplificationmethodologies that can also be utilized to increase the copy number oftarget nucleic acid. The polynucleotides detected in the method of theinvention can be further evaluated, detected, cloned, sequenced, and thelike, either in solution or after binding to a solid support, by anymethod usually applied to the detection of a specific nucleic acidsequence such as another polymerase chain reaction, oligomer restriction(Saiki et al., Bio/Technology 3:1008-1012 (1985)), allele-specificoligonucleotide (ASO) probe analysis (Conner et al., Proc. Natl. Acad.Sci. USA 80: 278 (1983), oligonucleotide ligation assays (OLAs)(Landegren et al., Science 241:1077 (1988)), RNAse Protection Assay andthe like. Molecular techniques for DNA analysis have been reviewed(Landegren et al, Science 242: 229-237 (1988)). Following DNAamplification, the reaction product may be detected by Southern blotanalysis, without using radioactive probes. In such a process, forexample, a small sample of DNA containing the polynucleotides obtainedfrom the tissue or subject are amplified, and analyzed via a Southernblotting technique. The use of non-radioactive probes or labels isfacilitated by the high level of the amplified signal. In one embodimentof the invention, one nucleoside triphosphate is radioactively labeled,thereby allowing direct visualization of the amplification product byautoradiography. In another embodiment, amplification primers arefluorescently labeled and run through an electrophoresis system.Visualization of amplified products is by laser detection followed bycomputer assisted graphic display, without a radioactive signal.

The methods of the present invention can involve a real-timequantitative PCR assay, such as a Taqman® assay (Holland et al., ProcNatl Acad Sci USA, 88(16):7276 (1991)). The assays can be performed onan instrument designed to perform such assays, for example thoseavailable from Applied Biosystems (Foster City, Calif.). Primers andprobes for such an assay can be designed according to known proceduresin the art. For example, primers and probes for some of thedifferentially expressed melanoma skin markers of the present inventioncan include, but are not limited to, the following:

Endothelin 2 Forward CTGCCAAGGCGCTGTCA (SEQ ID NO:5) (Target:TGACAGCGCCTTGGCAG) (SEQ ID NO:6) Reverse TCAGTCCAGGGCCTTCGA (SEQ IDNO:7) (Target: TCGAAGGCCCTGGACTGA) (SEQ ID NO:8) Probe TGCCAGGGACCCC(SEQ ID NO:9) (Target: GGGGTCCCTGGCA) (SEQ ID NO:10) Inhibin (Activin)Forward AACATGCTGCACTTGAAGAAGAGA (SEQ ID NO:11) (Target:TCTCTTCTTCAAGTGCAGCATGTT) (SEQ ID NO:12) Reverse GAAGCTTTCTGATCGCGTTCA(SEQ ID NO:13) (Target: TGAACGCGATCAGAAAGCTTC) (SEQ ID NO:14) ProbeCCGGCTGGGTGACAT (SEQ ID NO:15) (Target: ATGTCACCCAGCCGG) (SEQ ID NO:16)InsulinBindingProtein7 Forward GCGTGTGCGTGTGCAAGA (SEQ ID NO:17)(Target: TCTTGCACACGCACACGC) (SEQ ID NO:18) Reverse CAGCCGCTCGGGTAGGT(SEQ ID NO:19) (Target: ACCTACCCGAGCGGCTG) (SEQ ID NO:20) ProbeCGCTGCCGCACAC (SEQ ID NO:21) (Target: GTGTGCGGCAGCG) (SEQ ID NO:22)IL1R1 Forward GCACAAGCCATATTTAAGCAGAAAC (SEQ ID NO:23) (Target:GTTTCTGCTTAAATATGGCTTGTGC) (SEQ ID NO:24) ReverseAACTCCATATAAGGGCACACAAGTC (SEQ ID NO:25) (Target:GACTTGTGTGCCCTTATATGGAGTT( (SEQ ID NO:26) Probe CTCCGTCTCCTGCAAC (SEQ IDNO:27) (Target: GTTGCAGGAGACGGAG) (SEQ ID NO:28)

Another non-limiting example of primers and probes of the presentinvention, are primers and probes that selectively hybridize to thetarget sequences identified above (SEQ ID NOS: 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, and 28).

Simple visualization of a gel containing the separated products may beutilized to analyze the melanoma skin markers according to the methodsof the present invention. For example, staining of a gel to visualizeseparated polynucleotides, a number of stains are well known to thoseskilled in the art. However, other methods known to those skilled in theart may also be used, for example scanning densitometry, computer aidedscanning and quantitation as well as others.

The method for detecting one or more melanoma skin markers mayalternatively employ the detection of a polypeptide product of one ofthese genes. The method for detecting a polypeptide derived from amelanoma skin marker in cells is useful for detecting melanoma bymeasuring the level of one or more melanoma skin marker product, forexample IL-1 RI, endothelin-2, ephrin-A5, IGF Binding Protein 7,HLA-A0202 heavy chain, Activin A (βA subunit), TNF RII, SPC4, and CNTFRα, in cells obtained from a subject suspected of having, or at risk ofhaving malignant melanoma. The levels of such melanoma skin markerproducts are indicative of malignant melanoma when compared to a normalor standard polypeptide profiles in a similar tissue. Thus, theexpression pattern of a melanoma skin marker product will vary dependingupon the presence and stage of malignant melanoma.

In this regard, the sample, as described herein, can be used as a sourceto isolate polypeptides. Measurement of a particular polypeptide, forexample IL-1 RI, endothelin-2, ephrin-A5, IGF Binding Protein 7,HLA-A0202 heavy chain, Activin A (βA subunit), TNF RII, SPC4, and CNTFRα, can serve as a method of detecting, staging, diagnosing, monitoring,prognosing, or otherwise assisting in management of malignant melanoma.For example, following skin scraping or skin stripping, using themethods described above, cells isolated from the stratum corneum may belysed by any number of means, and polypeptides obtained from the cells.These polypeptides may then be quantified using methods known to thoseof skill in the art, for example by ELISA.

Monoclonal antibodies to a particular polypeptide, for example, exampleIL-1 RI, endothelin-2, ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavychain, Activin A (βA subunit), TNF RII, SPC4, and CNTF Rα, can be usedin immunoassays, such as in liquid phase or bound to a solid phasecarrier, to detect polypeptide associated with a disorder, such asdermatitis. In addition, the monoclonal antibodies in these immunoassayscan be detectably labeled in various ways. Examples of types ofimmunoassays that can utilize monoclonal antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of thepolypeptide antigens using the monoclonal antibodies of the inventioncan be done utilizing immunoassays, which are run in either the forward,reverse, or simultaneous modes, including immunohistochemical assays onphysiological samples. Those of skill in the art will know, or canreadily discern, other immunoassay formats without undueexperimentation. In addition, there are a number of commerciallyavailable antibodies to cytokines of interest.

The term “immunometric assay” or “sandwich immunoassay” includessimultaneous sandwich, forward sandwich and reverse sandwichimmunoassays. These terms are well understood by those skilled in theart. Those of skill will also appreciate that antibodies according tothe present invention will be useful in other variations and forms ofassays which are presently known or which may be developed in thefuture. These are intended to be included within the scope of thepresent invention.

Monoclonal antibodies can be bound to many different carriers and usedto detect the presence of a cytokine polypeptide. Examples of well-knowncarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such using routineexperimentation. A cytokine polypeptide may be detected by themonoclonal antibodies when present in biological fluids and tissues suchas a skin sample.

In performing the assays it may be desirable to include certain“blockers” in the incubation medium (usually added with the labeledsoluble antibody). The “blockers” are added to assure that non-specificproteins, proteases, or anti-heterophilic immunoglobulins toanti-cytokine immunoglobulins present in the experimental sample do notcross-link or destroy the antibodies on the solid phase support, or theradiolabeled indicator antibody, to yield false positive or falsenegative results. The selection of “blockers” therefore may addsubstantially to the specificity of the assays.

It has been found that a number of nonrelevant (i.e., nonspecific)antibodies of the same class or subclass (isotype) as those used in theassays (e.g., IgG1, IgG2a, IgM, etc.) can be used as “blockers”. Theconcentration of the “blockers” (normally 1-100 μg/μl) may be important,in order to maintain the proper sensitivity yet inhibit any unwantedinterference by mutually occurring cross reactive proteins in thespecimen.

“Cytokine” as used herein means any number of factors that play a rolein cellular regulation or differentiation. For example, cytokines caninclude the family of interleukins (IL) including IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-14 as well as factors belongingto the transforming growth factor beta (TGF-β) superfamily, GM-CSF andinterferon.

As used herein, the term “biological factor” means a number of factorsthat have biological activity or play a biological role. For example,biological factor includes polynucleotides, such as DNA, RNA, mRNA andcDNA, polypeptides, such as IL-4, IL-8, and IL-13 proteins and fragmentsthereof, as well as lipids such as cholesterol, fatty acids, andinflammatory mediators such as leukotrienes, prostaglandins and others.

In another aspect, the present invention provides a non-invasive methodfor monitoring a suspicious lesion of a subject. The method includesanalyzing expression of one or more melanoma skin markers in a skinsample taken from the suspicious lesion at a first time point and asecond time point, and comparing the expression at the first time pointand the second time point. The melanoma skin markers include IL-1 RI,endothelin-2, ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavy chain,Activin A (βA subunit), TNF RII, SPC4, and CNTF Rα. Expression of themelanoma skin markers is related to melanoma, such that a change in theexpression of one or more of the melanoma skin markers over time isindicative of melanoma. In certain preferred embodiments, the skinsample includes nucleic acids, and is a human skin sample.

Time points can include any interval of time, but are typically at least2 weeks, and more typically at least 1 month apart. For certainembodiments, time points are 2 months, 3 months, 6 months, 1 year, or 2years apart. Samples can be taken at any number of time points,including 2, 3, 4, 5, etc. time points. Comparison of expressionanalysis data from different time points can be performed using any ofthe known statistical methods for comparing data points to assessdifferences in the data, including time-based statistical methods suchas control charting. Melanoma can be identified in the time series, forexample, by comparing expression levels to a cut-off value, or bycomparing changes in expression levels to determine whether they exceeda cut-off change value, such as a percent change cut-off value.

In another aspect, the present invention provides a non-invasive methodfor detecting expression of IL-1 RI, endothelin-2, ephrin-A5, IGFBinding Protein 7, HLA-A0202 heavy chain, Activin A (βA subunit), TNFRII, SPC4, and CNTF Rα in a skin sample. The method includes obtaining askin sample by applying an adhesive surface to the skin and removing theadhesive surface from the skin such that a skin sample comprisingnucleic acid in an amount sufficient for subsequent detection adheres tothe adhesive surface after its removal. Then, analyzing expression ofIL-1 RI, endothelin-2, ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavychain, Activin A (βA subunit), TNF RII, SPC4, and CNTF Rα in the nucleicacids of the skin sample. As disclosed herein, this method is useful fordetecting, diagnosing, staging, monitoring, and managing malignantmelanoma.

In another embodiment the invention provides a kit for detectingmalignant melanoma. The kit includes one or more detection reagents, forexample oligonucleotide primers or probes that are complementary to apolynucleotide sequence encoding at least one of IL-1 RI, endothelin-2,ephrin-A5, IGF Binding Protein 7, HLA-A0202 heavy chain, Activin A (βAsubunit), TNF RII, SPC4, and CNTF Rα. The oligonucleotide primers can bespotted on a bioarray which is provided in the kit. The kit can includea skin sample collection device and probes that selectively bind to IL-1RI, endothelin-2, and ephrin-A5. Such a kit may also include a carriermeans being compartmentalized to receive in close confinement one ormore containers such as vials, tubes, and the like, each of thecontainers comprising one of the separate elements to be used in themethod. If present, a second container may comprise a lysis buffer. Thekit can alternatively include a computer-type chip on which the lysis ofthe cell will be achieved by means of an electric current.

The kit, in preferred embodiments, includes a skin sample collectiondevice such as a rigid instrument capable of removing the epidermallayer of the skin (e.g. a sterile #15 scalpel), and/or an adhesivesurface, such as an adhesive tape, for example, Duct tape (333 Ducttape, Nashua tape products), Scotch® tape (3M Scotch 810, St. Paul,Minn.), or a similar product. A preferred adhesive tape is D-SQUAME®(CuDerm, Dallas, Tex.). The kit can also include a cell lysis buffersuitable for preserving nucleic acids in the skin sample.

The kit can also have containers containing probes or primers foramplification of or hybridization to the target nucleic acid sequencewhich may or may not be labeled, or a container comprising a reporter,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradionuclide label. The term “detectably labeled deoxyribonucleotide”refers to a deoxyribonucleotides that is associated with a detectablelabel for detecting the deoxyribonucleotide. For example, the detectablelabel may be a radiolabeled nucleotide or a small molecule covalentlybound to the nucleotide where the small molecule is recognized by awell-characterized large molecule. Examples of these small molecules arebiotin, which is bound by avidin, and thyroxin, which is bound byanti-thyroxin antibody. Other methods of labeling are known to those ofordinary skill in the art, including enzymatic, fluorescent compounds,chemiluminescent compounds, phosphorescent compounds, and bioluminescentcompounds.

The kit can include one or more primer pairs, including a forward primerthat selectively binds upstream of a melanoma skin marker gene on onestrand, and a reverse primer, that selectively binds upstream of amelanoma skin marker gene on a complementary strand. The melanoma skinmarker genes are typically one or more of Interleukin-1 RI (IL-1 RI),endothelin-2, ephrin-A5, Insulin-like Growth Factor (IGF) BindingProtein 7, Human Leukocyte Antigen (HLA)-A0202 heavy chain, Activin A(βA subunit), Tumor Necrosis Factor (TNF) RII, SPC4, and CiliaryNeurotrophic Factor (CNTF) Rα. Primer pairs according to this aspect ofthe invention are typically useful for amplifying a polynucleotide thatcorresponds to a melanoma skin marker genes using amplification methodsdescribed herein.

In another aspect the invention provides a method of screening forcompounds or identifying compounds which may cause or prevent malignantmelanoma, or which may be used to treat malignant melanoma. In thisaspect, for example, cells of the skin, such as epidermal cells,including keratinocytes and melanocytes, or dermal cells, such asfibroblasts, are contacted with a test compound under conditions whichwould induce malignant melanoma formation. The expression of melanomaskin markers is then detected.

The conditions under which contact is made are variable and will dependupon the type of compound, the type and amount of cells in the skin tobe tested, the concentration of the compound in the sample to be tested,as well as the time of exposure to the compound. The skill in the art indetermining the proper conditions under which a compound may causemelanoma are known and would require only routine experimentation, ifany. The skin cells may be isolated using the techniques describedabove, e.g. by scraping or tape stripping, the cells may then be exposedto the test compound in vitro. Alternatively, cultured skin cells orskin constructs may be used. For example, skin cells may be culturedfrom any source under standard cell culture conditions on a solid orsemi-solid support until they become sufficiently confluent. Uponconfluence or subconfluence the cells are then exposed to the testcompound. Polynucleotides are then isolated from the cells which havebeen exposed to the compound and quantitated as described above.

For example, and not by way of limitation, skin cells can be isolated bythe tape or scraping method above and mRNA isolated. The mRNA can thenbe quantified using the probes for particular melanoma skin markers.Alternatively, the mRNA may be amplified by RT-PCR prior to detection ofthe polynucleotide. As described above, quantitation of a polynucleotidederived from a melanoma skin marker, can be used to detect, diagnose,stage, screen, and assist in management, of malignant melanoma.

The present invention is not to be limited in scope by the specificexamples provided for below, which are intended as single illustrationsof individual aspects of the invention and functionally equivalentmethods and components are within the scope of the invention.

EXAMPLE 1 Non-Invasive Recovery of Sub-Stratum Corneum Cells A. RecoveryUsing a Rigid Surface

Skin cells can be recovered non-invasively by scraping the skin with asterile # 15 scalpel. The scalpel is held at an angle approximately 15degrees from horizontal and repeatedly but gently scraped across an areaof skin that is approximately 1×1 cm in size. The epidermal cells aretransferred to a sterile tissue culture well by scraping the bladeagainst the interior wall of the well. When the glistening epidermallayer is reached, the scraping is stopped prior to causing any bleeding,to avoid contaminating the scraping(s) with blood. The cells aredeposited in a sterile 1 cm petri dish and about 300 ml of lysis bufferis added to the culture well. The lysis buffer is pipetted up and downuntil the epidermal cells are completely lysed.

RNA lysis buffer is added within 10 minutes of initiation of thescraping. The sterile tissue culture well is maintained on dry ice. Thecells are dissolved in the RNA lysis buffer, transferred into RNAse freecentrifuge tubes and the total RNA is extracted.

B. Recovery Using an Adhesive Surface

Skin cells can be recovered non-invasively by using Duct tape (333 Ducttape, Nashua tape products), Scotch® tape (3M Scotch® 8 10, St. Paul,Minn.), D-SQUAME® (CuDerm, Dallas, Tex.), or a similar product. The skinis stripped up to a maximum 25 times. Additionally, it will berecognized that the stickier the tape, the fewer strippings arerequired. The skin cells were recovered by vortexing and thencentrifuging the tape in an RNAse-free Eppendorf tube containing lysisbuffer. The same lysis buffer was reused for each piece of tape used ata single skin site. The entire procedure was performed in less than 90minutes. The process of tape stripping itself does not affect the skincytokine profile during the first few hours after the procedure is done.Moreover, during the early hours after stripping no inflammatory cellsmigrate from the circulation into the dermis or epidermis.

RNA was immediately extracted from cells adhering to the strip byvigorously vortexing the tape in 0.5 ml TriReagent (Molecular ResearchCenter, Inc., Cincinnati, Ohio). Yeast transfer RNA (4 μg) was thenadded as carrier RNA before the total RNA was isolated and purifiedaccording to the manufacturer's instructions. The total isolated RNAfrom each sample was used in an RNAse protection assay (RiboQuant®Multiprobe RNAse Protection Assay System, PharMingen, Inc., San Diego,Calif.) without prior measurement of the amount of RNA by ODmeasurement. Assays were performed with samples on standard acrylamidesequencing gels and used to identify digested cytokine messages. Gelscontaining digested RNA bands were first exposed to a Phosphor Screen(Molecular Dynamics, Inc., Sunnyvale, Calif.). The exposed screen wasthen scanned with a phosphorimager Storm 860 (Molecular Dynamics, Inc.).Intensities of bands in each sample were analyzed with the softwareImageQuant™ (Molecular Dynamics, Inc.).

Appropriate care should be taken to prevent RNAse contamination of thesamples since skin is a rich source of RNAse that can quickly degradeRNA released from damaged epidermal cells. The sample collection andextraction techniques described herein demonstrate that skin RNA canindeed be obtained without significant degradation as indicated by theability to detect mRNA by RPA.

EXAMPLE 2 Analysis of Cells Obtained by Tape Stripping

Irritant contact dermatitis (ICD) was induced by applying 0.5% sodiumlauryl sulfate (SLS) in distilled water for 72 hours to the upper arm.After this exposure, the erythema was graded according to standardscoring sales (Fisher's Contact Dermatitis. 4th ed. Rietschel, R. L. andFowler, J. F. Jr. eds. Williams & Wilkins, Baltimore, 1995, pg. 29).Allergic contact dermatitis (ACD) was induced by applying dibutylsquarate in acetone to the upper arm of the same subject under occlusionfor 48 hours. The upper arms of the same individual (subject #1) weretape stripped 12 times and processed as described in Example 2 above.

FIG. 1, lane 1 shows the RNA isolated from an ACD erythematous area ofskin, read clinically as 3+ erythema, that was induced by squarate. Lane3 is the RNA from ICD erythematous skin, clinically scored as 2+erythema, induced after exposure to 0.5% SLS. After exposure of thex-ray film, the band for cytokine IL-4 can be clearly seen in lane 1,but not in lane 3 which contains RNA from ICD cells. Thus, the cytokinepattern in the ACD reaction clearly differed from the ICD reaction andnormal skin seen in lane 2.

In a subsequent experiment, all subjects with dermatitis had mRNAencoding the cytokine IL-4 in cells from skin in areas that haddemonstrated an ACD reaction (lanes 8, 11, 13 in FIG. 2). By contrast,IL-4 was not visible in any of the ICD treated areas of skin or innormal skin samples obtained from the same subjects. Furthermore, in 4of 5 subjects (subjects 2, 3, 4 and 5 in FIG. 2), IL-8 was present inerythematous areas of skin, whether the erythema was induced by anirritant or an allergic reaction, but not in the RNA obtained fromnormal skin. Thus, IL-8 mRNA was generically indicative of dermatitis.

The mRNA encoding IL-13, a cytokine secreted by activated T cells, waspresent in 3 of the 4 erythematous areas of skin (lanes 5, 8, 11, 13 inFIG. 2) in which allergic inflammation had been induced by squarate. Afaint band could be seen in the approximate area(s) expected to containthe mRNA with the molecular weight associated with gamma interferon(IFN-γ) (lanes 8 and 11 in FIG. 2). These bands were present in the mRNAextracted from 2 of the 5 squarate (ACD) treated skin samples. As wasthe case for IL-13, the tentative band for IFN-γ mRNA was seen in thesame lanes that also had mRNA for IL-4.

IL-14, a B cell growth factor, was present in some of the squaratetreated skin samples as well as some of the SLS treated skin samples(FIG. 2). IL-9, a multifunctional cytokine, was detected in all 13samples that could be visualized in this experiment. In addition, themRNA for the inducible isoform of nitric oxide synthase (iNOS) and IL-9were seen in every lane that could be visualized clearly (13 of 15samples) (FIG. 2). The presence of IL-4 in the same lanes as IL-13strongly suggests that these two cytokine markers were induced by anallergic reaction in the skin from which the samples were obtained.

The clinical quantification of the erythema visualized in the variousskin reactions is documented in Tables 1 and 2.

TABLE 1 ACD REACTIONS SKIN SUBJECT REACTION IL-4 IL-8 IL-9 IL-13 iNOSIFNγ #1 0 ND ND + ND + ND #2 2+ + + NT NT NT NT #3 2+ + + + + + + #42+ + + * + * + #5 2+ + + + + + + ND = not detected * gel not readable NT= not tested 2+ = moderate erythema (red)

TABLE 2 ICD REACTIONS SKIN SUBJECT REACTION IL-4 IL-8 IL-9 IL-13 iNOSIFNγ #1 0 ND ND + ND + ND #2 2+ + + NT NT NT NT #3 1+ ND + + + + ND #41+ (low) ND ND + ND ND ND #5 3+ * + * * * * ND = not detected * gel notreadable NT = not tested 1+ = mild erythema (pinkish) 2+ = moderateerythema (red) 3+ = strong erythema (beet red)

EXAMPLE 3

To further examine the relationship between the cytokines and the degreeof inflammation in subject numbers 3-5, the IL-4, IL-8 and IL-13 RNAlevels were normalized to the corresponding housekeeping gene levels(Table 3). Among the three subjects analyzed, a correlation existsbetween the RNA levels and the severity of the reactions. Table 2 showsthat the samples from the strongest skin reactions were also the onesthat demonstrated the largest relative amount of IL-8 in the ACDreaction. For example, subject #4 with a 2+ reaction at the ACD site andonly a slight (low +1) reaction at the ICD site showed an approximatetwo fold difference in the IL-8/GAPDH ratios when comparing the ICD andACD reactions using the RPA method described above. In addition, onewould predict an ACD reaction if, on the gel, there is a band for IL-4and a value for IL-4/GAPDH of about 0.001 or higher. Also, an ACDreaction can be confirmed where there is an IL-13 band with anIL-13/GAPDH value of about 0.13 or higher (Table 3).

TABLE 3 TYPE OF REACTION Subject ICD ACD IL-4/GAPDH NC NC 3 IL-8/GAPDH0.3495 0.8867 iNOS/GAPDH 0.2202 0.2652 IL-13/GAPDH 0.070 0.251IL-4/GAPDH 0 0.01559 4 IL-8/GAPDH 0.2879 0.61080 iNOS/GAPDH 0.071070.2661 IL-13/GAPDH 0.117 0.134 IL-4/GAPDH 0 0.07255 5 IL-8/GAPDH 0.25411.3023 iNOS/GAPDH 0.05315 0.1951 IL-13/GAPDH 0.055 0.158 NC = notcalculated

A number of embodiments of the present invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLE 4

This example identifies genes which are differentially expressed at themRNA level, in early or late stage melanoma cells compared to normalcells. These genes are melanoma skin markers according to the presentinvention. An adhesive tape was used to obtain mRNA from frozen tissuesamples of malignant melanoma and control nevi, and skin surface geneexpression profiling studies (SSGEP) with a cDNA array were performed,to produce a set of potential genetic markers that can be utilized for anon-invasive molecular diagnostic screening procedure for melanoma.Candidate genes that were identified on the cDNA array were furtherinvestigated using RT-PCR with SYB-R green detection. The genes encodingIL-1 RI, endothelin-2, and ephrin-A5 were found to be differentiallyregulated between control nevi and early melanoma using the SSGEPprocedure on frozen tissue samples. Likewise, the genes encoding IGFBinding Protein 7, HLA-A0202 heavy chain, Activin A (βA subunit), TNFRII, SPC4, and CNTF Rα were found to be differentially regulated betweencontrol nevi and advanced melanoma. Identification of genes implicatedin the progression of early stage melanoma is important for developingmolecular diagnostic assays for screening and staging melanoma, as wellas for developing more effective therapeutics for this deadly disease.

Since melanoma appears to modulate gene expression of neighboring cells,it was hypothesized that it may be possible to detect early melanoma byexamining gene expression profiles of outer skin cells overlying asuspicious lesion. Neighboring effects are most obvious and welldocumented for various secreted cytokines. For example, it has beenpreviously described for the expression of macrophage migratoryinhibitory factor, MIF, that it stimulates the expression ofmetalloproteinases in fibroblasts, which may help to invade the dermisin case of a malignant tumor that excessively produces MIF (Onodera S,et al., J. Biol. Chem., 275:444-450, 2000). MIF was found by ourcoworkers to be overexpressed in highly tumorigenic variants of melanomacell lines if compared to less tumorigenic variants (Vogt et al.,Submitted, 2001). Similar neighboring effects may happen in thekeratinocyte population, too: In the case of melanoma detection, basalcells that were formerly in the vicinity of a melanocyte, and havemigrated into the upper epidermis through the differentiation process,may preserve mRNA profiles induced in response to the underlyingmelanoma, and paracrine effects of a plethora of pathological cytokinesmay induce a characteristic profile of the keratinocytes adjacent to thetumor and higher upwards in the epidermis. This is reflected by theclinical finding, that advanced tumors often lead to atrophy and erosionof the epidermis overlaying the lesion.

Molecular analysis of cells in their native environment is thought toprovide the most accurate picture of the in vivo disease state (Liottaand Petricoin, Nature Reviews/Genetics 1:48-56, 2000). Since biopsiedtissues are three dimensional structures containing numerous interactingcellular subpopulations, it is difficult to isolate the molecularsignatures of a particular cell type. The present Example utilizes amethod for obtaining a sample from the surface of a suspected melanomalesion or a control nevi.

In order to develop a non-invasive molecular diagnostic assay for earlymelanoma detection, the use of a skin surface sampling method wascombined with a specialized cDNA array to determine differentiallyexpressed genes between malignant melanoma and normal nevi. The genearray contained approximately 600 genes, specific for cytokines,receptors, and growth factors. An adhesive tape was utilized to obtainskin cells. This technique is referred to herein as skin surface geneexpression profiling (SSGEP).

In SSGEP, epidermal cells are removed using a tape stripping methodologyin which successive tapes are applied to the same skin site, and removedin a rapid progression, until a sufficient depth is reached for samplinggene expression profiles (need tape stripping reference). The epidermisconsists predominantly of keratinocytes (>90%), which differentiate fromthe basal layer, moving outward through various layers having decreasinglevels of cellular organization, to become the cornified cells of thestratum corneum layer. Renewal of the epidermis occurs every 20-30 daysin normal skin. Other cell types present in the epidermis includemelanocytes, Langerhans cells, and Merkel cells.

Since melanoma appears to modulate gene expression of neighboring cells,it was hypothesized that it may be possible to detect early melanoma byexamining gene expression profiles of outer skin cells overlying asuspicious lesion. Neighboring effects are most obvious and welldocumented for various secreted cytokines. For example, it has beenpreviously described for the expression of macrophage migratoryinhibitory factor, MIF, that it stimulates the expression ofmetalloproteinases in fibroblasts, which may help to invade the dermisin case of a malignant tumor that excessively produces MIF (Onodera etal., J. Biol. Chem. 275:444-450, 2000). MIF was found by our coworkersto be overexpressed in highly tumorigenic variants of melanoma celllines if compared to less tumorigenic variants (Vogt et al., Submitted,2001). Similar neighboring effects may happen in the keratinocytepopulation, too. In the case of melanoma detection, basal cells thatwere formerly in the vicinity of a melanocyte, and have migrated intothe upper epidermis through the differentiation process, may preservemRNA profiles induced in response to the underlying melanoma, andparacrine effects of a plethora of pathological cytokines may induce acharacteristic profile of the keratinocytes adjacent to the tumor andhigher upwards in the epidermis. This is reflected by the clinicalfinding, that advanced tumors often lead to atrophy and erosion of theepidermis overlaying the lesion.

Materials and Methods Tape Stripping

Frozen tissue samples for gene expression profiling studies of malignantmelanoma were obtained from the University of Regensburg Department ofDermatology. A group of six control benign nevi, four early malignantmelanoma samples (Pagetoid epidermal scattering), and 4 advancedmalignant melanoma samples (epidermal atrophy) were randomly selectedfrom the tissue collection. D-squame tapes (Cu-Derm, Texas) wereutilized to isolate skin cells from the frozen biopsies for skin surfacegene expression profiling. The D-squames were cleaned with RNA zap(Ambion) prior to use. The surface of the biopsy samples was cleanedwith alcohol, dried, and then moistened with a 1/10 dilution ofcommercial RNAse inhibitor. Isolation of skin cells from the frozenbiopsy samples was performed in a cryostat by pressing a D-squame tapetightly against the tissue, quickly removing the tape, and repeatingthis tape-stripping procedure until the tape surface was covered withcells. Up to 30 tapes were collected in this manner from a singlebiopsy, and placed in a sterile RNase free culture dish. The D-Squametape samples were stored at −80° C. until the next processing step.

RNA Extraction

D-squame tape samples were brought to −20 in the cryostat. For RNAextraction, tapes from a single biopsy sample were successivelysubmersed in one volume (750 μl) of RLT lysis buffer (Qiagen) in amicrocentrifuge tube by placing the tapes individually in themicrocentrifuge tube containing the RLT buffer, performing a rapidfreeze-thaw cycle in liquid nitrogen, and scraping the tape surface witha sterile inoculation loop. After extraction, each tape was removedusing forceps and discarded. The procedure was repeated until all tapesin the pool were extracted. RNA was isolated following the RNeasyprotocol, with on column DNAase digest. RNA was eluted from the columnusing 30 μl of RNAse free water (Promega), and concentrated to about 5μl at 4° C. using a Speed Vic.

Synthesis of cDNA

Double-stranded cDNA was synthesized by reverse transcription of thetotal RNA and subsequent amplification of the first strand cDNA usingthe SMART PCR II kit (Clontech). The optimum number of PCR cycles wasdetermined by taking aliquots from the PCR reaction after 10, 15, 20,and 25 reaction cycles, and analyzing by gel electrophoresis. The laneon the gel having a moderately strong smear of cDNA ranging from 0.5 to6 kb, and several bright bands, was chosen to represent the optimumnumber of PCR cycles for an abundance of cDNA transcripts. A secondreaction was prepared and cycled to this optimal number of cycles. TheQiagen PCR purification kit was utilized to purify the double strandedcDNA (Becker et al., J. Invest. Dermatol. 116:983-988, 2001).

Probe Labeling

Approximately 25 ng of the double stranded cDNA from the PCR reactionwas labeled with α ³²P-deoxycytidine triphosphate using randomly primedKlenow fragment synthesis. Non-incorporated nucleotides were removedusing the Nuctrap and beta shield kits (Stratagene).

Array Hybridization

The Sigma Panorama array, containing 600 cytokines, receptors, andgrowth factors was used to probe for differentially expressed genes inthe malignant melanoma biopsies (Sigma-Genosys, The Woodlands, Tex.).The Sigma Panorama filter array was prehybridized for 24 hours in 5 mLof Sigma-Genosys hybridization solution at 65 C. The ³²P-labeled cDNAsamples (probes) were denatured for 2 minutes in a boiling water bath,and then applied to the filter array for 48 hours at 65 C. The filterarray was washed three times with a 0.5×SSPE/1% SDS solution (RTsolution I) for 3 minutes, and once with RT Solution II at 65 C for 20minutes (0.1×SSPE/1% SDS). The array filter was exposed toautoradiography film for 24-48 hours and to phosphor imager cassettesfor quantitation.

Quantitation of Hybridization Signals

The hybridization signals were measured by a phosphor imager, andanalyzed using the AIDA Metrix® software (Raytest). A backgroundcorrection was performed, and the signals were normalized using thesignals from the housekeeping genes. For each group of malignantmelanoma samples (early and late stage), pair-wise comparisons were madebetween the malignant melanoma samples and the normal nevi controlsamples based on the normalized integrated signal intensities. Signalsfor candidate genes differing by more than a factor of three wereidentified, and statistical significance was determined using anon-parametric test (U test).

RT-PCR Confirmation of Markers

Real time RT-PCR with SyBr Green detection was utilized to verify IL1-RIresults in a semi-quantitative manner, the relative abundance of thecandidate marker genes with respect to the control gene GAPDH. Forexample, for IL1 RI expression analysis using RT-PCR, the primers of SEQID NO: 1 and SEQ ID NO:3 were used.

Results

By comparing skin surface gene expression profiles in early malignantmelanoma with control nevi, three differentially expressed genes wereidentified (Table 4). For advanced malignant melanoma, sixdifferentially expressed genes were identified by comparison with thecontrol nevi (Table 5). Each gene identified in a malignant melanomagroup (early or advanced) was differentially expressed in all four ofthe melanoma samples. The differentially expressed genes in the earlymalignant melanoma group were distinct from those in the advanced group.

TABLE 4 Markers discriminating early melanomas from nevi. Endothelin-2 p= 0.005 ephrin-A5 p = 0.009 IL-1 RI p = 0.009

TABLE 5 Markers discriminating advanced melanomas from nevi. HLA-A 0201heavy chain p = 0.002 Activin A (bA subunit) p = 0.002 IGF BindingProtein 7 p = 0.003 TNF RII p = 0.03 SPC4 p = 0.03 CNTF Ra p = 0.03

Expression levels for all of the advanced stage melanoma skin markersidentified herein, in benign nevi versus advanced malignant melanoma areshown in FIG. 3. The expression levels of all of the advanced stagemelanoma skin markers were higher in advanced malignant melanoma samplesthan in benign nevi.

Expression levels for all of the early stage melanoma skin markersidentified herein, in benign nevi versus advanced malignant melanoma areshown in FIG. 4. The expression levels of all of endothelin-2 andephrinA5 were higher in early stage malignant melanoma samples than inbenign nevi. The expression level of IL1 RI was lower in early stagemelanoma than in benign nevi.

Semi-quantitative RT PCR of IL1 RI gene expression, was performed on aset of tape sampled material different from that used for the experimentdescribed above. Among 11 patient tape samples, IL1 RI gene expressionwas lower in melanoma samples than in benign samples, except for onefalse negative (i.e. malignant melanoma patient sample with an elevatedIL1 RI reading), and one false positive reading (i.e. benign nevipatient sample with a depressed IL1 RI reading) (FIG. 5).

2. Discussion

Over 100 genes have been reported to have significance in theprogression of malignant melanoma (see Table 6). A few potentiallypromising candidates under discussion, P16, CDKN2A, melastatin, and theMage-A family of genes are among those reported to have potentialdiagnostic or prognostic value.

While germline mutations in p16 or CDKN2A are found in a significantpercentage of relatively rare melanoma families, p16 mutations are rarein sporadic tumors (Bataille, Clin. Exp. Dermatol. 25(6):464-467, 2000).Deletion or inactivation of CDKN2A appears to be involved in theprogression rather than the initiation of sporadic malignant melanoma(Cachia et al., Clin. Cancer Res. 6(9):3511-3515, 2000).

Based on in-situ hybridization studies with biopsy samples, it wasrecently reported that melastatin mRNA expression appeared to correlatewith melanocytic tumor progression, melanoma tumor thickness, and thepotential for melanoma metastasis (Deeds et al., Human Pathology31(11):1346-1356, 2000; Duncan et al., Journal of Clinical Oncology19(2):568-576, 2001). The loss of melastatin mRNA expression correlatedwith poor prognosis. Downregulation of the melastatin gene wasoriginally observed using a mouse melanoma cell line, B16, with highmetastatic potential (Duncan et al., J Clin Oncol, 19(2):568-76, 2001).Since the melastatin marker appears to be melanocyte specific, aprognostic assay based on melastin mRNA will most likely require abiopsy.

A genetic test based on three members of a family of cancer relatedgenes called MAGE-A was recently reported for the detection ofmetastatic cancer in blood or tissue (Miyashiro et al., ClinicalChemistry 47(3):505-512, 2001). A multiplex assay is advantageous, sincegene markers are often expressed at a low frequency, and variations ingene expression profiles exist across individuals. While 40% of thepatients with stage IV melanoma had detectable levels of Mage-A mRNAs intheir blood samples, 0% of stage I patients, and 14% of stage II orstage III patients exhibited detectable Mage-A mRNAs.

Using a hierarchical clustering algorithm, it was recently shown thatdifferentially regulated genes in melanoma biopsy samples were relatedto a vascular-like morphology that correlated with motility andinvasiveness (Bittner et al., Nature 406:536-540, 2000). However, theclustering pattern could not be correlated with clinical or histologicalfindings.

This example identifies three genes, IL-1 RI, endothelin-2, andephrin-A5, as being differentially regulated in early melanoma (stage I,pT1) using a non-invasive skin sampling method. Although endothelin-1and endothelin-3 have been previously linked to melanoma progression, anassociation between elevated levels of ephrin-2 and early melanoma hasnot been reported.

Although IL-1 RI has been reported to be linked to melanoma progression,these reports were based on cultured melanoma cells and were not skincells from sample obtained using a non-invasive sampling method (Dekkeret al., Melanoma Res. 7(3):223-230, 1997). Furthermore, Dekkar et al.1997, is silent as to differential expression of IL-1 RI during aparticular stage (i.e. early stage) of melanoma progression.

Ephrin-A5 has been reported to modulate cell adhesion and morphology(Robbins, EMBO J. 19(20):5396-5405, 2000). The ephrins are ligands forthe Eph receptors, which are the largest known subfamily of receptortyrosine kinases. ephrins are cell surface-associated proteins importantfor development, particularly in cell-cell interactions that promoteprocesses such as nervous system patterning, angiogenesis, andoncogenesis.

Up-regulation of ephrin-A1 has been previously observed in melanomabiopsies, in increasing amounts in more advanced melanomas (Easty etal., Int. J. Cancer 84:494, 1999). Since the pro-inflammatory cytokinesTNF-α and IL-1β both induce ephrin-A1 expression in melanoma cells, itwas postulated that ephrin-A1 expression may be related to hostinflammatory responses to advanced lesions. ephrin-A1 was also suspectedto so promote vascularization of the tumor, thus contributing to furthergrowth and metastasis (Easty et al., Int. J. Cancer 84:494, 1999). It islikely that ephrin-A5 contributes to melanoma progression through asimilar mechanism.

The development of a molecular diagnostic screening test for earlymelanoma comprising the markers IL-1 RI, endothelin-2, and ephrin-A5, incombination with a non-invasive skin surface sampling method isdescribed here. While the samples described here were obtained fromfrozen tissue, gene expression profiles are expected to be identical infreshly isolated human skin cell samples on the adhesive tape becausethe tissues used were snap frozen in Nitrogen immediately after excisionand kept at −80° C. until the study was performed. Skin samples isolatedon tape appear to be stable toward RNA degradation due to the relativelydesiccated nature of stratum corneum cells. The markers identified herehave utility in both diagnostic and prognostic applications and serve asaids for initial therapeutic decisions possibly saving many lives andreducing the cost of this safety by avoiding unnecessary excisions andbiopsies.

TABLE 6 Actin AX/α/β/acidic Adenosin Deaminase B2m BAGE BAX bcl-2 BFGFbrn-2/N-Oct3 c-jun, c-fos, jun-B c-k-ras c-kit c-met c-myc c-ski c-src-1Calcyclin Calmodulin Cathepsin B,D Cyclin A,B,D CDK4 E-Cadherin ECKEndothelin-1 Rezeptor EWS-ATF-1 FGF-Rezeptor-1 Sialyltransferase FamilieGM-CSF gro/MGSA H/Ki/K/N ras H-2Db/H-2Kb (MHC Klasse I) HER2/neu(Rezeptortyrosinkinase) HOX Gene (Homeoboxgene) FGF Familie hst ICAM1IGF-1 IL1,2,6,8,10 IL4-Rezeptor Integrine z.B. αVβ3 Interferon SystemMHC Klasse II Kollagenase (Gelatinase A/B, Metalloproteinasen)rap1-Krev1 Laminine Lerk Familie (Liganden der eph-verwandtenRezeptortyrosinkinasen) LRP M-CSF MAGE1/2 MCAF MCP-1 mda-6 (p21), mda-7MDR-1 Me14-D12 ME20 ME491 Mel-18 Melan-A Methallothionein MIAMitochondriale Gene MnSOD MRP MSH-R p15 (MTS2) MUC18/MCAM MYB MZ2-E,Fn-myc NF-1 Nma Nmb NME (nm23-1/2) NSE Ornithindecaboxylasep-Glycoproteine p16INK4 (CDKN2/MTS1) p97 PAIs PCNA PDGF PKC isoformenpp125FAK PRB Ribosomale Gene S100 TAL1 TCL5 Tenascin TGF α TGF β 1,2,3TGF β II Rezeptor Thrombospondin Thymosin β 10 TIMP-1 TNF α TPATransglutaminase-TT Tropomyosin 3 uPA-Rezeptoren VEGF-R VinculinVitronectin-Rezeptor VLA-1,2,4,5 WT1

1. A non-invasive method for detecting malignant melanoma in a skinsample of a subject, the method comprising analyzing expression in theskin sample of one or more melanoma skin markers selected fromInterleukin-1 RI (IL-1 RI), endothelin-2, ephrin-A5, Insulin-like GrowthFactor (IGF) Binding Protein 7, Human Leukocyte Antigen (HLA)-A0202heavy chain, Activin A (A subunit), Tumor Necrosis Factor (TNF) RII,SPC4, and Ciliary Neurotrophic Factor (CNTF) Rα, wherein expression ofthe melanoma skin markers is related to melanoma, thereby detectingmelanoma in the skin sample.
 2. The method of claim 1, wherein the skinsample is obtained by applying at least one application of an adhesiveto the skin and removing the adhesive from the skin, or scraping theskin with an instrument, to remove a sample comprising a nucleic acidfrom the skin.
 3. The method of claim 1, wherein expression is analyzedfor at least one of IL-1 RI, endothelin-2, and ephrin-A5, and wherein adecrease in IL-1 RI expression levels, or an increase in endothelin-2 orephrin-A5 expression levels is indicative of melanoma.
 4. The method ofclaim 1, wherein expression is analyzed for IL-1 RI, endothelin-2, andephrin-A5, and wherein a decrease in IL-1 RI expression levels, and anincrease in endothelin-2 or ephrin-A5 expression levels is indicative ofearly stage melanoma.
 5. The method of claim 1, wherein expression isanalyzed for one or more of IGF Binding Protein 7, HLA-A0202 heavychain, Activin A (βA subunit), TNF RII, SPC4, and CNTF Ra wherein anincrease in expression levels of the one or more melanoma skin markersis indicative of melanoma.
 6. The method of claim 1, wherein expressionis analyzed for IGF Binding Protein 7, HLA-A0202 heavy chain, Activin A(βA subunit), TNF RII, SPC4, and CNTF Ra wherein an increase inexpression levels of the melanoma skin markers is indicative ofmelanoma.
 7. The method of claim 2, wherein the sample is obtained in amanner such that the skin nucleic acid profile after application is notaffected for up to about two hours.
 8. The method of claim 2, whereinthe skin sample is isolated by applying the adhesive surface to the skinbetween one and two times to obtain the skin sample.
 9. The method ofclaim 2, wherein the adhesive surface comprises an adhesive tape and theskin sample is isolated from a lesion suspected to be melanoma.
 10. Themethod of claim 2, wherein the skin sample is isolated by scraping anouter layer of skin with a rigid instrument and the skin sample isisolated from a lesion suspected to be melanoma.
 11. The method of claim2, wherein the nucleic acid comprises RNA, and expression is analyzed byanalyzing RNA levels of one or more of the melanoma skin markers. 12.The method of claim 11, wherein the RNA comprises mRNA, and expressionis analyzed by analyzing mRNA levels of one or more of the melanoma skinmarkers.
 13. The method of claim 1, wherein the skin sample comprisesnucleic acids, and wherein expression of the melanoma skin markers isanalyzed by detecting polynucleotides encoding melanoma skin markers.14. The method of claim 1, wherein the skin sample comprises nucleicacids, wherein expression of the melanoma skin markers is analyzed bymeasuring the amount of polynucleotides encoding the melanoma skinmarkers, and wherein an increase or a decrease in expression of themelanoma skin markers is indicative of malignant melanoma.
 15. Themethod of claim 2, wherein expression of the melanoma skin markers isanalyzed by applying the nucleic acids to a bioarray.
 16. The method ofclaim 1, wherein the skin sample is a human skin sample isolated from alesion suspected to be melanoma.
 17. The method of claim 1, whereinexpression of IL-1 RI, endothelin-2, ephrin-A5, IGF Binding Protein 7,HLA-A0202 heavy chain, Activin A (βA subunit), TNF RII, SPC4, and CNTFRα is analyzed in the nucleic acid sample, wherein a decrease inexpression levels of IL-1 RI is related to early stage melanoma, anincrease in expression levels of endothelin-2 and ephrin-A5 are relatedto early stage melanoma, and an increase in expression levels of IGFBinding Protein 7, HLA-A0202 heavy chain, Activin A (βA subunit), TNFRII, SPC4, and CNTF Rα is related to late stage melanoma.
 18. The methodof claim 1, wherein expression is analyzed for at least one ofendothelin-2 and ephrin-A5.
 19. A non-invasive method for stagingmalignant melanoma in a skin sample from a human subject, the methodcomprising analyzing expression in the skin sample of one or moremelanoma skin markers selected from Interleukin-1 RI (IL-1 RI),endothelin-2, ephrin-A5, Insulin-like Growth Factor (IGF) BindingProtein 7, Human Leukocyte Antigen (HLA)-A0202 heavy chain, Activin A(βA subunit), Tumor Necrosis Factor (TNF) RII, SPC4, and CiliaryNeurotrophic Factor (CNTF) Rα, wherein expression of the melanoma skinmarkers is related to a stage of melanoma, thereby staging melanoma inthe skin sample.
 20. The method of claim 19, wherein the nucleic acidsample is obtained by applying at least one application of an adhesiveto the skin and removing the adhesive from the skin, or scraping theskin with an instrument to remove a sample comprising a nucleic acidfrom the skin.
 21. The method of claim 19, wherein expression isanalyzed for at least one of IL-1 RI, endothelin-2, and ephrin-A5, andwherein a decrease in expression levels of IL-1 RI is indicative ofearly stage melanoma, and an increase in expression levels ofendothelin-2 and ephrin-A5 is indicative of early stage melanoma. 22.The method of claim 19, wherein expression is analyzed for IL-1 RI,endothelin-2, and ephrin-A5, and wherein a decrease in expression levelsof IL-1 RI and an increase in expression levels of endothelin-2 andephrin-A5 is indicative of early stage melanoma.
 23. The method of claim19, wherein expression is analyzed for one or more of IGF BindingProtein 7, HLA-A0202 heavy chain, Activin A (βA subunit), TNF RII, SPC4,and CNTF Ra and wherein increased expression of the one or more melanomaskin marker is indicative of late-stage melanoma.
 24. The method ofclaim 19, wherein expression is detected for IGF Binding Protein 7,HLA-A0202 heavy chain, Activin A (βA subunit), TNF RII, SPC4, and CNTFRα, and wherein increased expression of the melanoma skin markers isindicative of late-stage melanoma.
 25. The method of claim 19, whereinexpression is analyzed IL-1 RI, endothelin-2, ephrin-A5, IGF BindingProtein 7, HLA-A0202 heavy chain, Activin A (βA subunit), TNF RII, SPC4,and CNTF Rα in the nucleic acid sample, wherein a decrease in expressionlevels of IL-1 RI and an increase in expression levels of endothelin-2and ephrin-A5 is indicative of early stage melanoma and an increase inexpression of IGF Binding Protein 7, HLA-A0202 heavy chain, Activin A(βA subunit), TNF RII, SPC4, and CNTF Rα is indicative of late stagemelanoma.
 26. A non-invasive method for staging malignant melanoma in askin sample of a human subject, the method comprising a) obtaining askin sample by applying an adhesive surface to the skin and removing theadhesive surface from the skin such that a skin sample comprisingnucleic acid in an amount sufficient for subsequent detection adheres tothe adhesive surface after its removal; and b) analyzing expression inthe nucleic acid of melanoma skin markers selected from Interleukin-1 RI(IL-1 RI), endothelin-2, ephrin-A5, Insulin-like Growth Factor (IGF)Binding Protein 7, Human Leukocyte Antigen (HLA)-A0202 heavy chain,Activin A (βA subunit), Tumor Necrosis Factor (TNF) RII, SPC4, andCiliary Neurotrophic Factor (CNTF) Rα in the nucleic acid sample,wherein a decrease in expression levels of IL-1 RI and an increase inexpression levels of endothelin-2 and ephrin-A5 is indicative of earlystage melanoma and an increase in expression of IGF Binding Protein 7,HLA-A0202 heavy chain, Activin A (βA subunit), TNF RII, SPC4, and CNTFRα is indicative of late stage melanoma.
 27. The method of claim 26,wherein the sample is obtained in a manner such that the skin nucleicacid profile after application is not affected for up to about twohours, thereby obtaining a skin sample for use in isolating or detectinga nucleic acid in a skin sample.